Deuterated Cyclosporine Analogs and Methods of Making the Same

ABSTRACT

Cyclosporine derivatives are disclosed which possess enhanced efficacy and reduced toxicity over naturally occurring and other presently known cyclosporins and cyclosporine derivatives. The cyclosporine derivatives of the present invention are produced by chemical and isotopic substitution of the cyclosporine A (CsA) molecule by: (1) Chemical substitution and optionally deuterium substitution of amino acid 1; and (2) deuterium substitution at key sites of metabolism of the cyclosporine A molecule such as amino acids 1, 4, 9. Also disclosed are methods of producing the cyclosporine derivatives and method of producing immunosuppression with reduced toxicity with the disclosed cyclosporine derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/280,666, filed Nov. 15, 2005, now U.S. Pat. No. 7,521,421, which is acontinuation of U.S. application Ser. No. 10/319,835, filed Dec. 16,2002, now abandoned, which is a continuation of U.S. application Ser.No. 09/634,945, filed Aug. 7, 2000, now U.S. Pat. No. 6,613,739, whichis a continuation of U.S. application Ser. No. 09/184,109, filed Nov. 2,1998, now abandoned, which is a continuation under 35 U.S.C. § 120 ofInternational Patent Application Serial No. PCT/IB98/01693, filed Oct.8, 1998, which claims benefit of U.S. Provisional Application No.60/061,360, filed Oct. 8, 1997. The disclosure of each of the aboveapplications is incorporated herein by reference in its entirety.

INTRODUCTION AND BACKGROUND

Cyclosporin derivatives of the present invention are disclosed whichpossess enhanced efficacy and reduced toxicity over naturally occurringand other presently known cyclosporins and cyclosporine derivatives. Thecyclosporin derivatives of the present invention are produced bychemical and isotopic substitution of the cyclosporine A (CsA) moleculeby:

1. Chemical substitution and optionally deuterium substitution of aminoacid 1; and

2. Deuterium substitution at key sites of metabolism of the cyclosporineA molecule such as amino acids 1,4,9. The cyclosporins are a family of,neutral, hydrophobic cyclic undecapeptides, containing a novelnine-carbon amino acid (MeBmt) at position 1 of the ring that exhibitpotent immunosuppressive, antiparasitic, fungicidal, and chronicanti-inflammatory properties. The naturally occurring members of thisfamily of structurally related compounds are produced by various fungiimperfecti. Cyclosporines A and C, are the major components.Cyclosporine A, which is discussed further below, is a particularlyimportant member of the cyclosporin family of compounds. Twenty fourminor metabolites, also oligopeptides, have been identified: Lawen etal., J. Antibiotics 42, 1283 (1989); Traber et al., Helv. Chim. Acta 70,13 (1987); Von Wartburg and Traber Prog. Med. Chem., 25, 1 (1988).Isolation of cyclosporines A and C, as well as the structure of A werereported by A. Ruegger et al., Helv. Chim. Acta 59, 1075 (1976); M.Dreyfuss et al., J. Appl. Microbial. 3, 125 (1976). Crystal andmolecular structures of the iodo derivative of A have been reported byT. J. Petcher et al., Helv. Chh. Acta 59, 1480 (1976). The structure ofC was reported by R Traber et al., ibid. 60, 1247 (1977). Production ofA and C has been reported by E. Harri et al., U.S. Pat. No. 4,117,118(1978 to Sandoz). Isolation, characterization and antifungal activity ofB, D, E, as well as the structures of A through D have been reported byR Traber et al., Helv. Chim. Acta 60, 1568 (1977). Isolation andstructures of E, F, G, H, I: eidem, ibid 65, 1655 (1982). Preparation of[2-Deutero-3-fluoro-D-Ala]⁸-CsA is disclosed by Patchett et al in GB2,206,199A which was published on Dec. 29, 1988.

Cyclosporin was discovered to be immunosuppressive when it was observedto suppress antibody production in mice during the screening of fungalextracts. Specifically, its suppressive effects appear to be related tothe inhibition of T-cell receptor-mediated activation events. Itaccomplishes this by interrupting calcium dependent signal transductionduring T-cell activation by inactivating calmodulin and cyclophilin, apeptidly propyl isomerase. It also inhibits lymphokine production byT-helper cells in vitro and arrests the development of mature CD8 andCD4 cells in the thymus. Other in vitro properties include inhibition ofIL-2 producing T-lymphocytes and cytotoxic T-lymphocytes, inhibition ofIL-2 released by activated T-cells, inhibition of resting T-lymphocytesin response to alloantigen and exogenous lymphocytes, inhibition of IL-1production, and inhibition of mitogen activation of IL-2 producingT-lymphocytes. Further evidence indicates that the above effects involvethe T-lymphocytes at the activation and maturation stages.

Stimulation of TCR (T cell receptor) by foreign antigen on a majorhistocompatibility (MHC) molecule on the surface of the T cell resultsin the activation of a TCR signal transmission pathway (exact method oftransmission unknown) through the cytoplasm causing the signal resultsin the activation of nuclear transcription factors, i.e. nuclear factorof activated T-cells (NF-AT) which regulate transcription of T-cellactivation genes. These genes include that of lymphokine interleukin-2(IL-2). Translation of the message is followed by secretion of IL-2.T-cell activation also involves the appearance of the lymphokinereceptor IL-2R on the cell surface. After IL-2 binds to IL-2R, alymphokine receptor (LKR) signal transmission pathway is activated. Theimmunosuppressive drug, rapamycin, inhibits this pathway.

CsA is a potent inhibitor of TCR-mediated signal transduction pathway.It inhibits binding of NF-AT to the IL-2 enhancer, and thus inhibitstranscriptional activation. CsA binds to cyclophilin, which binds tocalcineurin, which is a key enzyme in the T-cell signal transductioncascade.

Cyclophilin is found in prokaryotic and eukaryotic organisms and isubiquitous and abundant. Cyclophilin is a single polypeptide chain with165 amino acid residues. It has a molecular mass of 17.8 kD. A roughlyspherical molecule with a radius of 17 angstroms, cyclophilin has aeight-stranded antiparallel beta barrel. Inside the barrel, the tightlypacked core contains mostly hydrophobic side chains. CsA has numeroushydrophobic side chains which allow it to fit into the cyclophilin betabarrel. Cyclophillin catalyzes the interconversion of the cis andtrans-rotamers of the peGIFdyl-prolyl amide bond of peptide and proteinsubstrates. Cyclophilin is identical in structure with peptidyl prolylcis-trans isomerase and beers structural resemblance to the superfamilyof proteins that transports ligands such as retinol-binding protein(RBP). These proteins carry the ligand in the barrel core. Butcyclophilin actually carries the ligand binding site on the outside ofthe barrel. The tetrapeptide ligand binds in a long deep groove on theprotein surface between one face of the beta barrel and theThr116-Gly130 loop.

Further properties have also been reported in studies of the biologicalactivity of CsA: J. F. Borel et al., Agents Actions 6, 468 (1976).Pharmacology: Eidem. Immunology 32, 1017 (1977); R. Y. Calne, Clin. Exp.Immunol. 35, 1 (1979). Human studies: R. Y. Calne et al., Lancet 2, 1323(1978); R. L. Powles et al., ibid. 1327; R. L. Powles et al., ibid 1,327 (1980). In vitro activity (porcine T-cells): D. J. White et al.,Transplantation 27, 55 (1979). Effects on human lymphoid and myeloidcells: M. Y. Gordon, J. W. Singer, Nature 279, 433 (1979). Clinicalstudy of CsA in graft-versus-host disease: P. J. Tutschka et al., Blood61, 318 (1983).

Mechanism of Cyclosporine A Action Cyclosporine A-Cyclophilin A Complex

CsA, as discussed above, binds to the cyclophilin beta barrel. ThirteenCyP A residues define the CsA binding site. These residues are Arg 55,Phe 60, Met 61, Gln 63, Gly 72, Ala 101, Asn 102, Ala 103, Gln 111, Phe113, Trp 121, Leu 122, His 126. The largest side-chain movements are 1.3A for Arg 55 and up to 0.7 A for Phe 60, Gln 63, and Trp 121. There arefour direct hydrogen bonds between the CyP A and CsA. Residues 4, 5, 6,7, 8 of CsA protrude out into the solvent and are thought to be involvedin binding the effector protein, calcineurin (Pflugl, G., Kallen, J.,Schirmer, T., Jansonius, J. N., Zurini, M. G. M., & Walkinshaw, M. D.(1993) Nature 361, 91-94.)

Function of CsA-CyP A Complex.

The CsA-CyP A complex inhibits the phosphatase activity of theheterodimeric protein serine/threonine phosphatase or calcineurin (Liu,J., Farmer, J. D., Lane, W. S., Friedman, L, Weissman, I., & Schreiber,S. L. (1991) Cell 66, 807-15.; Swanson, S. K., Born, T., Zydowsky, C.D., Cho, H., Chang, H. Y., & Walsh, C. T. (1992) Proc. Natl. Acad. Sci.USA 89, 3686-90). CyP A binds CsA with an affinity of ca 10 nM. Thecomplex is then presented to calcineurin (Liu, J., Farmer, J. D., Lane,W. S., Friedman, J., Weissman, I., & Schreiber, S. L. (1991) Cell 66,807-15.).

Calcineurin dephosphorylates the transcription factor NFAT found in thecytoplasm of T-cells. Dephosphorylation allows NFAT to translocate tothe nucleus, combine with jun/fos genes and activate the transcriptionof the IL-2 gene responsible for cell cycle progression, leading toimmune response. CsA-CyP A complex inhibits the phosphatase activity ofcalcineurin and ultimately immunosuppression (Etzkorn, F. A., Chang, Z,Stolz L. A., & Walsh, C. T. (1994) Biochemistry 33, 2380-2388.). NeitherCsA or CyP A alone are important immunologically. Only their complex isimportant (Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman,I., & Schreiber, S. L. (1991) Cell 66, 807-15).

Metabolism of Cyclosporine:

Cyclosporine is metabolized in liver, small intestine and kidney to morethan 30 metabolites. The structure of 13 metabolites and 2 phase IImetabolites have been identified and at least 23 further metaboliteshave been isolated by HPLC and their structures characterized by massspectrometry. The reactions involved in phase I metabolism ofcyclosporine are hydroxylation, demethylation as well as oxidation andcyclisation at amino acid 1. Several clinical studies and reports showedan association between blood concentrations of cyclosporine metabolitesand neuro- or nephrotoxicity. In vitro experiments indicate thatmetabolites are considerably less immunosuppressive and more toxic thanCsA.

As exemplified by the ever expanding list of indications for which CsAhas been found useful, the cyclosporin family of compounds find utilityin the prevention of rejection or organ and bone marrow transplants; andin the treatment of psoriasis, and a number of autoimmune disorders suchas type 1 diabetes mellitus, multiple sclerosis, auto immune uveitis,and rheumatoid arthritis. Additional indications are discussed infra.

As is generally accepted by those of skill in the art, inhibition ofsecretion of interleukin-2 (IL-2) and other lymphokines fromlymphocytes, is a useful indicator of intrinsic immunosuppressiveactivity of a cyclosporin analog. For a recent review of cyclosporinuses and mechanisms of action see Wenger et al Cyclosporine: Chemistry,Structure-Activity Relationships and Mode of Action, Progress inClinical Biochemistry and Medicine, vol. 2, 176 (1986).

Cyclosporin A is a cyclic peptide which contains several N-methyl aminoacids and, at position-8, contains a D-alanine. The structure ofCyclosporin A^(a) is given below: ^(a)Unless otherwise specified, eachof the amino acids of the disclosed cyclosporin is of theL-configuration.

As is the practice in the field, a particular cyclosporin analog may benamed using a shorthand notation identifying how the analog differs fromcyclosporin A. Thus, cyclosporin C which differs from cyclosporin A bythe threonine at position-2 may be identified as [Thr]² cyclosporin or[Thr]²CsA. Similarly, cyclosporin B is [Ala]²-CsA; cyclosporin D is[Val]²-CsA; cyclosporin E is [Val]¹¹ CsA; cyclosporin F is[3-DesoxyMeBmt]¹-CsA; cyclosporin G is [NVa]²-CsA, and cyclosporin H is[D-MeVal]¹¹-CsA.

D-Serine and D-Threonine have been introduced into the 8-position ofcyclosporin A by biosynthesis resulting in active compounds. See R.Traber et al. J. Antibiotics 42, 591 (1989). D-Chloroalanine has alsobeen introduced into position-8 of Cyclosporin A by biosynthesis. See A.Lawen et al J. Antibiotics 52, 1283 (1989).

Indications for Cyclosporine Therapy

Immunoregulatory abnormalities have been shown to exist in a widevariety of autoimmune and chronic inflammatory diseases, includingsystemic lupus erythematosis, chronic rheumatoid arthritis, type 1diabetes mellitus, inflammatory bowel disease, biliary cirrhosis,uveitis, multiple sclerosis and other disorders such as Crohn's disease,ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis,ichthyosis, and Graves ophthalmopathy. Although the underlyingpathogenesis of each of these conditions may be quite different, theyhave in common the appearance of a variety of autoantibodies andself-reactive lymphocytes. Such self-reactivity may be due, in part, toa loss of the homeostatic controls under which the normal immune systemoperates.

Similarly, following a bone marrow or an organ transplantation, the hostlymphocytes recognize the foreign tissue antigens and begin to produceantibodies which lead to graft rejection.

One end result of an autoimmune or a rejection process is tissuedestruction caused by inflammatory cells and the mediators they release.Anti-inflammatory agents, such as NSAID's (Non-SteroidalAnti-inflamatory Drugs), and corticosteroids act principally by blockingthe effect of or secretion of these mediators, but do nothing to modifythe immunologic basis of the disease. On the other hand, cytotoxicagents, such as cyclophosphamide, act in such a mm specific fashion thatboth the normal and autoimmune responses are shut off. Indeed, patientstreated with such nonspecific immunosuppressive agents are as likely tosuccumb to infection as they are to their autoimmune disease.

Generally, a cyclosporin such as cyclosporine A, is not cytotoxic normyelotoxic. It does not inhibit migration of monocytes nor does itinhibit granulocytes and macrophage action. Its action is specific andleaves most established immune responses intact. However, it isnephrotoxic and is known to cause the following undesirable sideeffects:

(1) abnormal liver function;(2) hirsutism;(3) gum hypertrophy;(4) tremor;(5) neurotoxicity;(6) hyperaesthesia; and(7) gastrointestinal discomfort.

A number of cyclosporins and analogs have been described in the patentliterature:

U.S. Pat. No. 4,108,985 issued to Ruegger, et al. on Aug. 22, 1978entitled, “Dihydrocyclosporin C”, discloses dihydrocyclosporin C, whichcan be produced by hydrogenation of cyclosporin C.

U.S. Pat. No. 4,117,118 issued to Harri, et al. on Sep. 26, 1978entitled, “Organic Compounds”, discloses cyclosporins A and B, and theproduction thereof by fermentation.

U.S. Pat. No. 4,210,581 issued to Ruegger, et al. on Jul. 1, 1980entitled, “Organic Compounds”, discloses cyclosporin C anddihydrocyclosporin C which can be produced by hydrogenation ofcyclosporin C.

U.S. Pat. No. 4,220,641, issued to Traber, et al. on Sep. 2, 1980entitled, “Organic Compounds”, discloses cyclosporin D,dihydrocyclosporin D, and isocyclosporin D.

U.S. Pat. No. 4,288,431 issued to Traber, et al. on Sep. 8, 1981entitled, “Cyclosporin Derivatives, Their Production and PharmaceuticalCompositions Containing Them”, discloses cyclosporin G,dihydrocylosporin G, and isocyclosporin G.

U.S. Pat. No. 4,289,851, issued to Traber, et al. on Sep. 15, 1981entitled “Process for Producing Cyclosporin Derivatives”, disclosescyclosporin D, dihyrocyclosporin D, and isocyclosporin D, and a processfor producing same.

U.S. Pat. No. 4,384,996, issued to Bollinger, et al. on May 24, 1983entitled “Novel Cyclosporins”, discloses cyclosporins having aβ-vinylene-α-amino acid residue at the 2-position and/or aβ-hydroxy-α-amino acid residue at the 8-position. The cyclosporinsdisclosed included either MeBmt or dihydro-MeBmt at the 1-position.

U.S. Pat. No. 4,396,542, issued to Wenger on Aug. 2, 1983 entitled,“Method for the Total Synthesis of Cyclosporins, Novel Cyclosporins andNovel Intermediates and Methods or their Production”, discloses thesynthesis of cyclosporins, wherein the residue at the 1-position iseither MeBmt, dihydro-MeBmt, and protected intermediates.

U.S. Pat. No. 4,639,434, issued to Wenger, et al on Jan. 27, 1987,entitled “Novel Cyclosporins”, discloses cyclosporins with substitutedresidues at positions 1, 2, 5 and 8.

U.S. Pat. No. 4,681,754, issued to Siegel on Jul. 21, 1987 entitled,“Counteracting Cyclosporin Organ Toxicity”, discloses methods of use ofcyclosporin comprising co-dergocrine.

U.S. Pat. No. 4,703,033 issued to Seebach on Oct. 27, 1987 entitled,“Novel Cyclosporins”, discloses cyclosporins with substituted residuesat positions 1, 2 and 3. The substitutions at position-3 include halogenH. Kobel and R. Traber, Directed Biosynthesis of Cyclosporins, EuropeanJ. Appln. Microbiol. Biotechnol., 14, 237B240 (1982), discloses thebiosynthesis of cyclosporins A, B, C, D & G by fermentation.

Additional cyclosporin analogs are disclosed in U.S. Pat. No. 4,798,823,issued to Witzel, entitled, New Cyclosporin Analogs with Modified “C-9amino acids”, which discloses cyclosporin analogs with sulfur-containingamino acids at position-1.

SUMMARY OF THE INVENTION

The present invention concerns chemically substituted and deuteratedanalogs of cyclosporine A and related cyclosporines.

An object of the present invention is to provide new cyclosporineanalogs which have enhanced efficacy and altered pharmacokinetic andpharmacodynamic parameters. Another object of the present invention isto provide a cyclosporine analog for the care of immunoregulatorydisorders and diseases, including the prevention, control and treatmentthereof. An additional object of the present invention is to providepharmaceutical compositions for administering to a patient in need ofthe treatment one or more of the active immunosuppressive agents of thepresent invention. Still a further object of this invention is toprovide a method of controlling graft rejection, autoimmune and chronicinflammatory diseases by administering a sufficient amount of one ormore of the novel immunosuppressive agents in a mammalian species inneed of such treatment. Finally, it is the object of this invention toprovide assess for the preparation of the active compounds of the pressinvention.

Substitution and deuteration of the cyclosporine molecule results inaltered physicochemical and pharmacokinetic properties which enhance itsusefulness in the treatment of transplantation rejection, host vs. graftdisease, graft vs. host disease, aplastic anemia, focal and segmentalglomerulosclerosis, myasthenia gravis, psoriatic arks, relapsingpolychondritis and ulcerative colitis.

Embodiments of the invention include CsA derivatives wherein one or morehydrogen atoms in the 1, 3 and 9 amino acid positions can be substitutedwith a deuterium atom and wherein the cyclosporine A derivatives areoptionally chemically substituted at the amino acid 9 position. Afurther specific embodiment of the invention is the CsA derivativerepresented by formula I:

where R is (i) a deuterium or (ii) a saturated or unsaturated straightor branched aliphatic chain of from 2 to 16 carbon atoms and optionallycon one or more deuterium atoms or an ester, ketone or alcohol of thecarbon chain and optionally containing one or more substituents selectedfrom halogen, nitro, amino, amido, aromatic, and heterocyclic, or (iii)R is an aromatic or heterocyclic group optionally containing a deuteriumatom, or (iv) R is a methyl group and X, Y, and Z are hydrogen ordeuterium provided that at least one of X, Y or Z is deuterium and R′ isan OH or an ester or is an O and together with a carbon adjacent to adouble bond on amino acid 1 form a heterocyclic ring such as 5-memberedrings where the heteroatom is oxygen. Other specific embodiments of thepresent invention include the CsA derivative of formula I where R is asaturated or unsaturated carbon chain of from 2 to 3 carbons containingone or more deuterium. Further specific embodiments include those offormulas 5g and 5e below:

DESCRIPTION OF THE FIGURES

FIG. 1 is the structure of cyclosporine A showing a site of deuterationat the amino acid 3 position.

FIG. 2 is the structure of cyclosporine A showing a site of deuterationat the amino acid 9 position.

FIG. 3 is scheme I of the synthesis of the cyclosporine derivatives.

FIG. 4 is scheme II of the synthesis of the cyclosporine derivatives.

DETAILED DESCRIPTION OF THE INVENTION

Substitution of deuterium for ordinary hydrogen and deuteratedsubstrates for protio metabolites can produce profound changes inbiosystems. Isotopically altered drugs have shown widely divergentpharmacological effects. Pettersen et al., found increased anti-cancereffect with deuterated 5,6-benzylidene-dl-L-ascorbic acid (Zilascorb)[Anticancer Res. 12, 33 (1992)].

Substitution of deuterium in methyl groups of cyclosporine will resultin a slower rate of oxidation of the C-D bond relative to the rate ofoxidation of a non-deuterium substituted C—H bond. The isotopic effectacts to reduce formation of demethylated metabolites and thereby altersthe pharmacokinetic parameters of the drug. Lower rates of oxidation,metabolism and clearance result in greater and more sustained biologicalactivity. Deuteration is targeted at various sites of the cyclosporinmolecule to increase the potency of drug, reduce toxicity of the drug,reduce the clearance of the pharmacologically active moiety and improvethe stability of the molecule.

Isotopic Substitution:

Stable isotopes (e.g., deuterium, ¹³C, ¹⁵N, ¹⁸O) are nonradioactiveisotopes which contain one additional neutron than the normally abundantisotope of the respective atom. Deuterated compounds have been used inpharmaceutical research to investigate the in vivo metabolic fate of thecompounds by evaluation of the mechanism of action and metabolic pathwayof the non deuterated parent compound. (Blake et al. J. Pharm. Sci. 64,3, 367-391, 1975). Such metabolic studies are important in the design ofsafe, effective therapeutic drugs, either because the in vivo activecompound administered to the patient or because the metabolites producedfrom the parent compound prove to be toxic or carcinogenic (Foster etal., Advances in Drug Research Vol. 14, pp. 2-36, Academic press,London, 1985).

Incorporation of a heavy atom particularly substitution of deuterium forhydrogen, can give rise to an isotope effect that could alter thepharmacokinetics of the drug. This effect is usually insignificant ifthe label is placed at a metabolically inert position of the molecule.

Stable isotope labeling of a drug can alter its physico-chemicalproperties such as pKa and lipid solubility. These changes may influencethe fate of the drug at different steps along its passage through thebody. Absorption, distribution, metabolism or excretion can be changed.Absorption and distribution are processes that depend primarily on themolecular size and the lipophilicity of the substance. These effects andalterations can affect the pharmacodynamic response of the drug moleculeif the isotopic substitution affects a region involved in aligand-receptor interaction.

Drug metabolism can give rise to large isotopic effect if the breakingof a chemical bond to a deuterium atom is the rate limiting step in theprocess. While some of the physical proper of a stable isotope-labeledmolecule are different from those of the unlabeled one, the chemical andbiological properties an the same, with one important exception: becauseof the increased mass of the heavy isotope, any bond involving the heavyisotope and another atom will be stronger than the same bond between thelight isotope and that atom. In any reaction in which the breaking ofthis bond is the rate limiting step, the reaction will proceed slowerfor the molecule with the heavy isotope due to “kinetic isotope effect”A reaction involving breaking a C-D bond can be up to 700 percent slowerthan a similar reaction involving breaking a C—H bond. If the C-D bondis not involved in any of the steps leading to the metabolite, there maynot be any effect to alter the behavior of the drug. If a deuterium isplaced at a site involved in the metabolism of a drug, an isotope effectwill be observed only if breaking of the C-D bond is the rate limitingstep There is evidence to suggest that whenever cleavage of an aliphaticC—H bond occurs, usually by oxidation catalyzed by a mixed-functionoxidase, replacement of the hydrogen by deuterium will lead toobservable isotope effect. It is also important to understand that theincorporation of deuterium at the site of metabolism slows its rate tothe point where another metabolite produced by attack at a carbon atomnot substituted by deuterium becomes the major pathway a process called“metabolic switching”. It is also observed that one of the mostimportant metabolic pathways of compounds containing aromatic systems ishydroxylation leading to a phenolic group in the 3 or 4 position tocarbon substituents. Although this pathway involves cleavage of the C—Hbond, it is often not accompanied by an isotope effect, because thecleavage of this bond mostly not involved in the rate limiting step. Thesubstitution of hydrogen by deuterium at the stereo center will induce agreater effect on the activity of the drug.

Synthesis of Cyclosporine Derivatives:

The staring material for the preparation of the compounds of thisinvention is cyclosporine A. The process for preparing the compounds ofthe present invention are illustrated as shown in scheme I in FIG. 3. Itwill be readily apparent to one of ordinary skill in the art reviewingthe synthetic route depicted below that other compounds with formula Ican be synthesized by substitution of appropriate reactants and agentsin the synthesis shown below.

The first step in the process for making deuterated cyclosporin analogsis the preparation of the key intermediate 3 and 6. This can be achievedby the oxidation of the double bond in the amino acid 1. Treatment ofcyclosporin with acetic anhydride and excess of dimethylaminopyridineprovided the hydroxyl protected acetyl cyclosporin. 2 Although cleavageof the double bond could then be accomplished by treatment of 2 withozone, or KMnO₄/NaIO₄, it was found out that OsO₄/NaIO₄ was the reagentof choice for the transformation to the aldehyde product 3. The reactionwas generally found to be cleaner, producing the required material andto proceed in higher yield. The drawback to this reaction is that OsO₄,is expensive and highly toxic, so that its use is limited. But theresults can be accomplished more economically by the use of H₂O, withOsO₄ present in catalytic amounts. t-butyl hydroxide in alkalinesolution and N-methylmorpholine-N-oxide can be substituted for H, inthis process. The aldehyde compound 3 was further treated with variousdeuterated alkyl or aryl triphenyl phosphonium derivatives (wittigreagents) and hydrolysis by alkaline solution provided the finalderivatives (5 a-h). We also developed a general procedure to obtainvarious compounds as shown in Scheme II in FIG. 4.

In this approach, the aldehyde derivative 3 a was treated with theWittig reagent prepared by using standard procedure. The resultantproduct on mild acid hydrolysis provided the key intermediate aldehydeproduct 6 This was further treated with second deuterated alkyl or aryltriphenylphosphonium halide reagents and on mild acid hydrolysis yieldedthe required products. This method provides control over the extensionof the diene system. By using this approach, olefinic double bonds canbe introduced step by step.

A third approach to prepare the deuterated compounds 5 a-h- is byheating non deuterated cyclosporin analogs described earlier, in adeuterated solvent such as deuterated water, deuterated acetic acid inthe presence of acid or base catalyst.

Preferred cyclosporins of the present invention include those whichcontain a deuterium and/or a chemical substitution on amino acid 1 suchas those of formula II:

Where X is

And R=—CHO, —CDO, —CH═CD-CD₃, CD=CD-CD₃, —CH═CH—CH═CD-CD₃,—CD=CH—CD=CD-CD₃, —CH═CH—CH═CD₂, —CD=CH—CD=CD₂, —CH═CD₂, —CH═CH₂ and—CD=CD₂, —CH═CH-CD₃, —CH═CH—CH═CH—CH₃, and —CH═CH—CH═CH₂. Otherpreferred embodiments of the invention include compounds where R ofabove formula (I) equals -D, —CHO, —CDO, —CD₃, —CH═CD-CD₃, —CD=CD-CD₃,—CH═CH—CH═CD-CD₃, —CD=CH—CD=CD-CD₃, —CH═CH—CH=CD₂, —CD=CH—CD=CD₂,—CH═CD₂, —CD=CD₂, —CH═CH₂, —CH═CH—CD₃, —CH═CH—CH₃, —CH═CH—CH═CH—CH₃, and—CH═CH—CH═CH₂.

EXAMPLES Example 1

To a stirred solution of cyclosporine 1 (1.01 g, 0.94 mmol) in aceticanhydride (20 mL) at room temperature was added DMAP (150 mg, 1.23 mmol,1.5 eq). After stirring overnight, the reaction mixture was partitionedbetween EtOAc (50 ml) and water (25 ml). The separated EtOAc layer wasthen washed with water (50 ml) and brine (50 mL), dried (MgSO₄) and thesolvent removed in vacuo to give the crude product as a glassy solid.Purification by flash chromatography through a short column of silica(2% MeOH/DCM) and lyophilisation from benzene yielded 2 (1.044 g, 0.84mmol, quant.) as a fluffy, colourless solid; [α]_(D) ²⁵ −305.7 (c. 0.3,CHCl₃); ν_(max) (CHCl₃ cast)/cm⁻¹ 3328m, 2963m, 1746m, 1627s, 1528m,1472m, 1233m; δ_(H) (600 MHz, C₆D₆) 8.73 (1H, d, J=9.5 Hz, NH), 8.30(1H, d, J=7.0 Hz, NH), 7.92 (1H, d, J=7.5 Hz, NH), 7.49 (1H, d, J=7.5Hz, NH), 6.05 (1H, d, J=11.5 Hz), 5.88 (1H, dd, J=3.5, 11.5 Hz), 5.82(1H, d, J=11.5 Hz), 5.65 (1H, dd, J=4.0, 12.0 Hz), 5.60 (1H, dd, J=3.5,12.5 Hz), 5.63-5.57 (1H, m), 5.51-5.45 (1H, m), 5.37 (1H, dd, J=5.5, 8.5Hz), 5.05-5.01 (2H, complex), 4.99 (1H, d, J=11.0 Hz), 4.76 (1H, p,J=7.0 Hz), 4.58 (1H, p, J=7.0 Hz), 4.02 (1H, d, J=13.5 Hz), 3.47 (3H,s), 3.30 (3H, s), 3.17 (3H, s), 3.11 (3H, s), 2.98 (3H, s), 2.68-2.62(1H, m), 2.63 (3H, s), 2.51-2.39 (2H, complex), 2.34-2.25 (8H, complex),2.03 (3H, s), 1.97-1.85 (2H, complex), 1.83 (3H, dd, J=1.0, 6.5 Hz),1.82-1.77 (2H, complex), 1.68-1.61 (3H, complex), 1.55 (3H, J=7.0 Hz),1.55-1.51 (1H, m), 1.44-1.38 (1H, m), 1.32-1.20 (5H, complex), 1.29 (3H,d, J=7.0 Hz), 1.21 (3H, d, J=6.5 Hz), 1.17 (3H, d, J=6.5 Hz), 1.14 (3H,d, J=6.5 Hz), 1.08 (3H, d, J=6.5 Hz), 1.04 (3H, d, J=6.0 Hz), 1.03 (3H,d, J=7.0 Hz), 1.00 (3H, d, J=7.0 Hz), 0.93 (3H, d, J=6.0 Hz), 0.92 (3H,d, J=6.5 Hz), 0.88-0.84 (9H, complex), 0.76 (3H, d, J=6.5 Hz), 0.57 (3H,d, J=6.5 Hz); δ_(C) (75 MHz, C₆D₆) 173.6, 173.2, 172.8, 172.6, 1713,171.1, 170.71, 170.67, 170.4, 1702, 169.8, 167.9 (C═O), 129.0, 126.2(C═C), 73.1 (COAc), 58.1, 57.1, 56.0, 55.0, 54.6, 54.2, 50.3, 49.9,48.6, 48.1, 47.8, 44.5, 40.8, 39.1, 35.7, 33.6, 32.9, 32.1, 31.5, 31.2,30.0, 29.7, 29.5, 29.3, 24.9, 24.6, 24.4, 24.0, 23.6, 23.4, 23.3, 21.7,21.1, 21.0, 20.6, 20.3, 19.5, 18.5, 18.0, 17.7, 17.5, 17.4, 14.9, 9.7;m/z (Electrospray)

Example 2

To a solution of compound 2 (289 mg, 0.23 mmol) in a 1:1 mixture ofdioxane and water (5 mL) was added firstly sodium metaperiodate (100 mg,0.47 mmol, 2 eq) and secondly a solution of osmium tetraoxide (5 mL; 0.5g, OsO₄ in 250 mL of solvent). Two-phase work-up, purification by flashcolumn chromatography (40% acetone in petroleum ether) andlyophilisation from benzene gave compound 3. (226 mg, 0.18 mmol, 80%) asa fluffy, colourless solid; [α]_(D) ²⁵ −260.0 (c. 0.1, CHCl₃); ν_(max)(CHCl₃ cast)/cm⁻¹ 3325m, 2962m, 1748w, 1724w, 1677m, 1626s, 1228m, 755m;δ_(H) (300 MHz, C₆D₆) 8.63 (1H, d, J=9.5 Hz, NH), 8.16 (1H, d, J=7.0 Hz,NH), 7.95 (1H, d, J=7.5 Hz, NH), 7.48 (1H, d, J=9.0 Hz, NH), 5.93 (1H,d, J=7.5 Hz), 5.84 (1H, dd, J=4.0, 11.5 Hz), 5.70 (1H, d, J=11.5 Hz),5.56-5.54 (1H, m), 5.32 (1H, dd, J=5.5, 8.0 Hz), 5.07-4.88 (3H,complex), 4.72 (1H, p, J=7.0 Hz), 4.49 (1H, p, J=7.0 Hz), 3.98 (1H, d,J=14.0 Hz), 3.42 (3H, s, CH ₃N), 3.27 (3H, s, CH ₃N), 3.12 (3H, s, CH₃N), 3.07 (3H, s, CH ₃N), 2.91 (3H, s, CH ₃N), 2.79 (3H, s, CH ₃N), 2.59(3H, s, CH ₃N), 2.42-2.08 (10H, complex), 1.94 (3H, s, CH ₃CO₂), 1.47(3H, d, J=7.0 Hz), 1.24 (3H, 7.0 Hz), 1.14-1.09 (9H, complex), 1.04 (3H,d, J=6.5 Hz), 1.01 (3H, d, J=7.0 Hz), 0.96 (3H, d, J=6.5 Hz), 0.92 (3H,d, J=6.5 Hz), 0.91 (3H, d, J=6.5 Hz), 0.89 (3H, d, J=6.0 Hz), 0.83 (6H,d, J=6.5 Hz), 0.74 (3H, d, J=6.5 Hz), 0.59 (3H, d, J=6.5 Hz); δ_(C) (75MHz, C₆D₆) 202.5 (CHO), 174.4, 174.0, 173.7, 172.8, 171.6, 171.5, 171.2,171.1, 170.6, 170.2, 1702, 168.1, 73.0, 58.7, 57.6, 56.7, 55.5, 55.0,54.5, 49.4, 48.9, 48.5, 48.1, 45.0, 44.6, 41.3, 39.8, 38.8, 37.7, 36.2,32.5, 32.0, 31.6, 30.9, 30.3, 30.0, 29.8, 29.6, 25.6, 25.3, 25.0, 24.8,24.5, 24.0, 23.8, 23.4, 22.0, 21.7, 21.2, 20.5, 20.0, 19.8, 18.8, 18.5,18.2, 17.4, 15.2, 10.0; m/z (Electrospray) 1232.8 (MH⁺, 100%).

Example 3

Method A: To a solution of compound 3 (315 mg, 0.26 mmol) in THF (5 mL)at 0° C. was added a solution of the deutero-phosphorus ylid (2.67 mmol,˜10 eq), prepared from d₅-ethyltriphenylphosphonium iodide. Afterwork-up, purification by flash column chromatography (30% to 60% acetonein PE) and HPLC (60% to 65% MeCN in water), then lyophilisation frombenzene yielded compound 4 (153 mg, 0.12 mmol, 47%) as a fluffy,colourless solid.

Method B: To a stirred solution of compound a (287 mg, 023 mmol) in THF(5 mL) under Ar at −78° C. was carefully added a solution of phosphorusylid (formed by the addition of sodium hexamethyldisilylamide (1.0M;2.25 mL, 2.25 mmol ˜10eq) to a suspension ofd₅-ethyltriphenylphosphonium iodide (480 mg, 1.13 mmol, ˜5 eq) in THF(10 mL) under Ar at room temperature). After stirring for 2 hr withgradual warming to room temperature, the reaction mixture was cooled to0° C. and was quenched by the addition of 10% AcOH/THF (10 mL). Thereaction mixture was concentrated in vacuo and partitioned between water(20 mL) and EtOAc (20 mL). The aqueous layer was further extracted withEtOAc (20 mL) and the combined organic extracts were then washed with 1NHCl (20 mL) and water (20 mL), dried (MgSO₄) and the solvent removed invacuo to give the crude product. Purification by flash columnchromatography (40% acetone in petroleum ether) and lyophilisation frombenzene yielded compound 4d (84 mg, 67 μmol, 29%) as a fluffy,colourless solid; [α]_(D) ²⁵ −283.0 (c. 0.1, CHCl₃); ν_(max) (CHCl₃cast)/cm⁻¹ 3320m, 3010m, 29595, 2924s, 2871m, 2853m, 1743m, 1626s, 756s;δ_(H) (600MHz, C₆D₆) 8.78 (1H, d, J=9.5 Hz), 8.33 (1H, d, J=7.0 Hz),7.99 (1H, d, J=7.5 Hz), 7.59 (1H, d, J=9.0 Hz), 6.09 (1H, d, J=11.5 Hz),5.92 (1H, dd, J=4.0, 11.0 Hz), 5.86 (1H, d, J=11.5 Hz), 5.72-5.64 (2H,complex), 5.62 (1H, dd, J=3.5, 12.5 Hz), 5.40 (1H, dd, J=5.5, 8.5 Hz),5.10-5.02 (3H, complex), 4.80 (1H, q, J=7.0 Hz), 4.60 (1H, q, J=7.0 Hz),4.05 (1H, d, J=14.0 Hz), 3.51 (3H, s), 3.31 (3H, s), 3.20 (3H, s), 3.13(3H, s), 3.01 (3H, s), 2.87 (3H, s), 2.64 (3H, s), 2.45 (1H, dt, J=4.0,12.5 Hz), 2.36-2.20 (10H, complex), 2.06 (3H, s), 1.93-1.79 (3H,complex); δ_(D) (84 MHz, C₆H₆) δ_(C) (125 MHz, C₆D₆) 174.5, 173.7,173.6, 173.1, 171.7, 171.4, 170.9, 170.7, 170.6, 170.3, 170.0, 168.4,130.2 (C═C), 123.8 (C═C), 73.8 (MeBmt C-3), 58.7, 58.1, 57.6, 57.1,55.5, 55.0, 54.5, 49.4, 49.0, 48.6, 48.2, 45.0, 41.4, 39.9, 39.0, 37.8,34.2, 33.9, 32.6, 32.3, 32.0, 31.4, 30.9, 30.8, 30.2, 30.1, 30.0, 29.9,29.8, 29.6, 28.5, 25.6, 25.3, 25.0, 24.9, 24.8, 24.1, 23.9, 23.8, 23.6,23.1, 22.1, 21.7, 21.4, 20.7, 20.0, 19.9, 19.8, 18.9, 18.7, 18.6, 18.3,17.4, 15.3, 14.3, 10.2; m/z (Electrospray) 1270 ([M+Na]⁺, 100%), 1286([M+K]⁺, 20).

Example 4

To a stirred solution of 4d (84 mg, 67 μmol) in MeOH (5 mL) and water(2.5 mL) at room temperature was added potassium carbonate (99 mg, 0.72mmol, ˜10 eq). After stirring overnight the MeOH was removed in vacuoand the aqueous residue was partitioned between EtOAc (10 mL) and 5%citric acid solution (10 mL). The EtOAc layer was then washed with water(10 mL) and brine (10 ml), dried (MgSO₄) and the solvent removed invacuo to give the crude product. HPLC purification (60% to 65% MeCN inwater) and lyophilisation from benzene yielded compound 5d (59 mg, 49μmol, 70%) as a fluffy, colourless solid; [α]_(D) ²⁵ −262.0 (c. 0.05,CHCl₃); ν_(max) (CHCl₃ cast)/cm⁻3318m, 3008m, 2960s, 2872m, 1627s,1519m, 1470m, 1411m, 1295m, 1095m, 754m; δ_(H) (600 MHz, C₆H₆) 8.27 (1H,d, J=9.5 Hz), 7.96 (1H, d, J=7.5 Hz), 7.63 (1H, d, J=8.0 Hz), 7.45 (1H,d, J=9.0 Hz), 5.87 (1H, dd, J=3.5, 11.0 Hz), 5.74 (1H, d, J=7.5 Hz),5.73-5.69 (1H, m), 5.66-5.64 (1H, br d, J=11.0 Hz), 5.79 (1H, dd, J=4.0,11.5 Hz), 3.39 (1H, dd, J=5.5, 10.5 Hz), 5.33 (1H, dd, J=5.5, 8.5 Hz),5.24 (1H, d, J=11.0 Hz), 5.12 (1H, dt, J=7.5, 10.0 Hz), 4.88-4.79 (3H,complex), 4.22 (1H, dd, J=5.5, 7.5 Hz), 4.00 (1H, d, 13.5 Hz), 3.72 (3H,s), 3.22 (3H, s), 3.06 (3H, s), 2.97 (3H, s), 2.92 (3H, s), 2.85 (3H,s), 2.67-2.60 (1H, m), 2.58 (3H, s), 2.56-2.50 (1H, br m), 2.33-2.23(4H, complex), 2.20-2.07 (4H, complex), 1.80-1.74 (3H, complex), 1.67(3H, d, J=7.0 Hz), 1.56-1.50 (2H, complex), 1.46-1.23 (9H, complex),1.17-1.13 (16H, complex), 1.06 (3H, d, J=6.5 Hz), 1.02 (3H, d, J=7.0Hz), 0.98 (3H, d, J=6.5 Hz), 0.96 (3H, d, J=7.0 Hz), 0.92-0.89 (9Hcomplex), 0.86 (3H, t, J=7.5 Hz), 0.83 (3H, d, J=6.0 Hz), 0.64 (3H, d,J=6.5 Hz); δ_(D) (84 MHz, C₆H₆) 1.64 (CD₃ ); δ_(C) (75 MHz, C₆H₆) 174.2,174.1, 174.0, 173.7, 171.8, 171.4, 171.2, 170.5, 170.4, 170.3, 169.8,130.2, 124.1, (99.2,) 74.3, (67.1,) 66.3, 66.1, 61.0, 59.5, 58.3, 57.8,55.7, 55.5, 55.4, 49.4, 49.0, 48.4, 45.3, 41.4, 39.6, 39.0, 37.8, 36.5,36.1, 35.8, 33.7, 31.6, 30.8, 30.4, 30.1, 29.9, 29.5, 29.4, 25.5, 25.2,25.0, 24.9, 24.5, 24.2, 23.8, 23.7, 23.6, 22.0, 21.4, 20.0, 18.8, 18.5,17.8, 16.0, 10.1; m/z (Electrospray) 1206 ([M+H]⁺, 30%), 1228 ([M+Na]⁺,100), 1244 ([M+K]⁺, 25).

Example 5

To a vigorously stirred mixture of compound 3 (49 mg, 39.8 μmol) anddeuterated d₃-allyltriphenylphosphonium bromide (311 mg, 812 μmol, ˜20eq) in benzene (3 mL) at room temperature was added 1N NaOH (3 mL).Stirring was continued at room temperature for 5 days, after which timethe 2 layers were separated, the benzene layer was washed with water (5mL), dried (MgSO₄) and the solvent removed in vacuo to give the crudeproduct. Purification by HPLC (20% to 60% MeCN in water) andlyophilisation from benzene yielded compound 4g (23 mg, 18.3 μmol, 47%)as a fluffy, colourless solid; [α]_(D) ²⁵ −2642 (c. 0.24, CHCl₃);ν_(max) (CHCl₃ cast)/cm⁻¹ 3322m, 2959m, 1744m, 1626s, 1231m, 754m; δ_(H)(300 MHz, C₆D₆ complex due to 1:1 ratio of geometrical isomers 8.73 (d,J=9.5 Hz, NH), 8.72 (d, J=9.5 Hz, NH), 8.29 (d, J=76.5 Hz, NH), 8.26 (d,J=6.5 Hz, NH), 7.92 (d, J=7.5 Hz, 7.86 (d, J=7.5 Hz, NH), 7.53 (d, J=9.0Hz, NH), 7.49 (d, J=9.0 Hz, NH), 7.10-6.70 (complex), 6.33 (br t, J=11.0Hz), 6.18 (d, J=10.5 Hz), 6.12 (d, J=10.5 Hz), 6.05 (d, J=11.0 Hz), 6.03(d, J=11.0 Hz), 5.90-5.53 (complex), 5.37 (dd, J=6.0, 8.0 Hz), 5.20 (d,J=12.0 Hz), 5.14 (d, J=12.0 Hz), 5.07-4.97 (complex), 4.80-4.70(complex), 4.57 (p, J=7.0 Hz), 4.02 (d, J=14.0 Hz), 4.01 (d, J=14.0 Hz),3.47 (s), 3.46 (s), 3.28 (s), 3.26 (s), 3.16 (3), 3.15 (s), 3.09 (s),2.97 (a), 2.96 (s), 2.84 (s), 2.62 (s), 2.48-2.23 (complex), 2.05 (s),2.03 (s), 1.95-1.59 (complex), 1.54 (d, J=7.0 Hz), 1.53-0.80 (complex),0.77 (d, J=6.5 Hz), 0.58 (d, J=6.5 Hz), 0.57 (d, J=6.5 Hz); δ_(C) (75MHz, C₆D₆) 174.5, 174.0, 173.9, 173.6, 173.5, 173.1, 171.7, 171.6,171.4, 170.9, 170.8, 170.6, 170.6, 1703, 169.8, 169.7, 168.4, 137.9,133.9, 133.5, 132.8, 132.3, 131.6, 130.1, 116.9, 115.0, 73.6, 58.6,57.6, 57.0, 56.8, 55.7, 55.5, 55.0, 54.9, 54.7, 54.5, 49.4, 48.9, 48.5,48.2, 48.1, 44.9, 41.5, 39.9, 39.0, 38.9, 37.8, 37.6, 36.6, 36.3, 34.1,33.7, 32.7, 32.1, 32.0, 31.5, 30.9, 30.7, 30.0, 29.8, 29.6, 25.6, 25.5,25.3, 25.2, 25.0, 24.9, 24.1, 23.9, 23.7, 23.6, 22.1, 21.7, 21.6, 21.4,21.3, 20.7, 20.0, 19.9, 18.9, 18.6, 18.3, 17.6, 15.3, 10.2; m/z(Electrospray) 1258.8 (MH⁺, 100%).

Example 6

To a vigorously stirred mixture of compound 3 (56 mg, 45.5 μmol) anddeuterated d₄-crotyltriphenylphosphonium bromide (360 mg, 907 μmol, ˜20eq) in benzene (3 mL) at room temperature a was added 1N NaOH (3 mL).Stirring was continued at room temperature for 5 days, after which timethe 2 layers were separated, the benzene layer was washed with water (5mL); dried (MgSO₄) and the solvent removed in vacuo to give the crudeproduct. Purification by HPLC (20% to 60% MeCN in water) andlyophilisation from benzene yielded compound 4e (23 mg, 18.1 μmol, 40%)as a fluffy, colourless solid; [α]_(D) ²⁵ 236.0 (c. 0.25, CHCl₃);ν_(max) (CHCl₃ cast)/cm⁻¹ 3324m, 2959m, 2871m, 1745w, 1626s, 1231m; N(300 MHz, C₆D₆) complex due to presence of 4 isomers 8.76 (d, J=6.0 Hz),8.73 (d, J=6.0 Hz), 829 (d, J=7.0 Hz), 7.93 (d, J=7.5 Hz), 7.88 (d,J=7.5 Hz), 7.53 (d, J=90.5 Hz), 7.62-7.31 (1H, complex), 7.16-6.88 (2H,complex), 6.59-6.39 (complex), 6.28 (t, J=11.0 Hz), 6.15 (d, J=10.5 Hz),6.09 (d, J=10 Hz), 6.05 (d, J=11.5 Hz), 6.03 (d, J=11.5 Hz) 5.90-5.82(complex), 5.68-5.35 (complex), 5.08-4.97 (complex), 4.81-4.72(complex), 4.63-4.53 (complex), 4.03 (d, J=14.0 Hz), 3.47 (s), 3.46 (s),3.28 (s), 326 (s), 3.17 (s), 3.15 (s) 3.09 (s), 2.98 (s), 2.97 (s), 2.83(s), 2.63 (s), 2.62 (s), 2.71-2.56 (complex), 2.47-2.23 (complex), 2.05(s), 2.04 (s), 2.03 (s), 2.02 (s), 1.98-0.82 (complex), 0.77 (d, J=6.5Hz), 0.58 (d, J=6.5 Hz), 0.58 (d, J=6.5 Hz); m/z (Electrospray) 1273.8

Example 7

To a stirred solution of compound 4g (20 mg, 15.9 μmol) in methanol (5mL) and water (1 mL) at room temperature was added potassium carbonate(30 mg, 217 μmol). After stirring overnight, the reaction mixture waspartitioned between EtOAc (10 mL) and 5% aqueous citric acid (10 mL).The aqueous layer was further extracted with EtOAc (5 mL), the combinedorganic layers were then washed with 5% citric acid (10 mL) and brine(10 mL), dried (MgSO₄) and the solvent removed in vacuo to give thecrude product. Purification by HPLC (65% MeCN) and lyophilisation frombenzene yielded compound 5g (10 mg, 8.2 μmol, 52%) as a fluffy,colourless solid; [α]_(D) ²⁵ −285.2 (c. 0.29, CHCl₃, ν_(max) (CHCl₃cast)/cm⁻¹ 3500-3200 br, 3319m, 2958m, 2927m, 1626s, 1520m, 1468m, 754m;δ_(H) (300 MHz, C₆D₆) complex due to the presence of 2 isomers 8.25 (d,J=10.0 Hz, NH), 8.13 (d, J=10.0 Hz, NH), 7.93 (d, J=7.0 Hz, NH), 7.84(d, J=7.0 Hz, NH), 7.67 (d, J=8.0 Hz, NH), 7.61 (d, J=8.0 Hz, NH), 7.55(d, J=8.5 Hz, NH), 7.54 (d, J=8.5 Hz, NH), 6.84 (t, J=10.5 Hz), 6.79 (t,J=10.5 Hz), 6.58 (t, J=10.5 Hz), 6.52 (t, J=10.5 Hz), 6.30-6.14(complex), 5.88-5.78 (complex), 5.75-5.66 (complex), 5.44-4.74(complex), 4.22-0.15 (complex), 3.95 (d, J=14.0 Hz), 3.93 (d, J=14.0Hz), 3.72 (s), 3.68 (s), 3.19 (s), 3.17 (s), 3.05 (s), 3.03 (s), 2.94(s), 2.93 (s), 2.89 (s) 2.86 (s), 2.82 (s), 2.81 (s), 2.72-2.53(complex), 2.55 (s), 2.54 (s), 2.49-2.36 (complex), 2.32-2.03 (complex),1.81-0.81 (complex), 0.65 (d, J=6.5 Hz)), m/z (Electrospray) 1216.8(MH⁺, 100%), 607.9 ([M+2H]²⁺, 15).

Example 8

To a stirred solution of compound 4e (18 mg, 14.2 μmol) in methanol (5mL) and water (1 mL) at room temperature was added potassium carbonate(35 mg, 254 μmol. After stirring overnight, reaction mixture waspartioned between EtOAc (10 mL) and 5% aqueous citric acid (10 mL). Theaqueous layer was further extracted with EtOAc (5 mL), the combinedorganic layers were then washed with 5% citric acid (10 mL) and brine(10 mL), dried (MgSO₄) and the solvent removed in vacuo to give thecrude product. Purification by HPLC (65% MeCN) and lyophilisation frombenzene yielded compound 5e (10 mg, 8.1 μmol, 57%) as a fluffy,colourless solid; [α]_(D) ²⁵ −285.5 (c. 0.11, CHCl₃); (300 MHz, C₆H₆)complex due to presence of 4 isomers 8.31 (d, J=9.5 Hz), 828 (d, J=95Hz), 8.16 (d, J=9.5 Hz), 8.14 (d, J=9.5 Hz), 7.96 (d, J=7.5 Hz), 7.95(d, J=7.5 Hz), 7.86 (d, J=7.5Hz), 7.85 (d, J=7.5 Hz), 7.63 (d, J=75H),7.59 (4 J 7.5 Hz), 7.50-7.44 (complex), 6.60-6.49 (complex), 6.32-6.11(complex), 5.88-5.83 (complex), 5.76-5.71 (complex),5.64-5.22-(complex), 5.17-5.08 (complex), 4.91-4.77 (complex), 4.26-4.18(complex), 3.99 (J=14.0 Hz), 3.97 (d, J=14.0 Hz), 3.74 (s), 3.73 (s),3.71 (s), 3.69 (s), 3.22 (s), 3.21 (s), 3.20 (s), 3.19 (s) 3.07 (s),3.06 (s), 3.05 (s), 2.97 (s), 2.96 (s), 2.95 (s), 2.92 (s), 2.91 (s),2.89 (s), 2.84 (s), 2.83 (s), 2.69-2.07 (complex), 2.58 (s), 2.57 (s),1.84-0.81 (complex), 0.64 (d, J=6.5 Hz); m/z (Electrospray) 1269.8([M+K]⁺, 5%), 1253.8 ([M+Na]⁺, 30), 1231.8 (MH⁺)

Example 9

The immunosuppressive activity can be tested for cyclosporine and thedisclosed cyclosporine analogs as described below. Calcineurin activityis assayed using a modification of the method previously described byFruman et al. (A Proc Natl Acad Sci USA, 1992). Whole blood lysates areevaluated for their ability to dephosphorylate a ³²P-labelled 19 aminoacid peptide substrate in the presence of okadaic acid, a phosphatasetype 1 and 2 inhibitor. Background phosphatase 2C activity (CsA andokadaic acid resistant activity) is determined and subtracted from eachsample, with the assay performed in the presence and absence of excessadded CsA. The man phosphatase activity is taken as calcineurinactivity.

Example 10

A mixed lymphocyte reaction (MLR) assay is performed with cyclosporineand the disclosed cyclosporine analogs. The MLR assay is useful foridentifying CsA derivatives with biological (immunosuppressive) activityand to quantify this activity relative to the immunosuppressive activityof the parent CsA molecule.

An example of a lymphocyte proliferation assay procedure useful for thispurpose is as follows:

-   1. Collect blood from two individuals (20 mls each) and isolate    lymphocytes using Ficoll-Paque (Pharmacia Biotech).-   2. Count lymphocytes at 1:10 dilution in 2% acetic acid (v/v).-   3. Prepare 10 mls of each lymphocyte populations (A+B) at 1×10⁶    cells/ml in DMEM/20% FCS (v/v).-   4. Set up a 96 well sterile tissue culture plate, flat bottom    (Sarstedt, at # 83.1835). To each well add:-   5. Aliquot 100 μl per well lymphocyte population A-   6. Aliquot 100 μl per well lymphocyte population B-   7. Aliquot 20 μl per well of drug (CSA and CSA derivatives) at 0,    2.5, 5, 10, 25, 50 and 100 μg/L in triplicate in DMEM with no    supplements.-   8. To measure the effect of drug on proliferation, incubate the    plate for 5 days at 37° C. in 5% CO₂ atmosphere-   9. On day 6, prepare 3.2 mls of 1:50 dilution of Methyl-³H-thymidine    (Amershame Life Science, cat #TRK 120) in DMEM with no supplements.    Add 30 μl per well and incubate for 18 hours at 37° C. in 5% CO₂    atmosphere-   10. On day 7 cells are harvested onto glass microfiber filters GF/A    (Whatman, cat #1820024) using a Cell-Harvestor (Skatron, cat    #11019). Wash cells 3× with 1.0 ml sterile distilled water.

Note: All procedures are done using sterile techniques in a biologicalflow hood.

-   11. Place filters in Scintillation vials and add 1.5 mls of    SciniSafe Plus 50% scintillation fluid (Fisher, cat #SX-25-5).-   12. Measure the amount of radioactivity incorporated in the    lymphocytes using a beta counter (Micromedic System Inc., TAURUS    Automatic Liquid Scintilation Counter) for 1.0 minute.-   13. Calculate averages and standard deviations for each drug and    express results as:

${\% \mspace{14mu} {Inhibition}} = {\left\lbrack {1 - \frac{{Ave}\mspace{14mu} {CPM}\mspace{14mu} {of}\mspace{14mu} {test}\mspace{14mu} {drug}}{{Ave}\mspace{14mu} {CPM}\mspace{14mu} {of}\mspace{14mu} {zero}\mspace{14mu} {drug}}} \right\rbrack \times 100}$%  Proliferation = 100 − %  Inhibition

From the results of the calcineurin assay and the mixed lymphocytereaction assay, it was found that cyclosporines that have beenchemically substituted and/or deuterated at the amino acid 1 positioncan possess significant immunosuppressant activity.

Example 11

Other cyclosporine derivatives of the invention which have been prepareinclude the following:

STRUCTURE CODE #

DB-b1-01

DB-b1-08

DB1-b1-11

DB1-b1-31

DB1-b1-45

DB-b186C

DB-b1-92b

DB-b1-93C

DB-b1-145D

DB-b1-147D

DB-01-148

DB-b1-151

DB-b1-176

DB-b1-179

DB-b1-180

DB-b1-192

DB-b1-193

DB-b1-134

DB-b1-194

DB-b1-195

DB-b1-196

DB-b1-50B

Drug Composition Formulation and Elicitation of Immunosuppression

Determination of the physicochemical, pharmacodynamic, toxicological andpharmcokinetic properties of the cyclosporine derivatives disclosed canbe made using standard chemical and biological assays and through theuse of mathematical modeling techniques which am known in the chemicaland pharmacological/toxicological arts. The therapeutic utility anddosing regimen can be extrapolated from the results of such techniquesand through the use of appropriate pharmacokinetic and/orpharmacodynamic models.

The compounds of this invention may be administered neat or with apharmaceutical carrier to a warm blooded animal in need thereof. Thepharmaceutical carrier may be solid or liquid.

This invention also relates to a method of treatment for patientssuffering from immunoregulatory abnormalities involving theadministration of the disclosed cyclosporines as the active constituent.

For the treatment of these conditions and diseases caused byimmunoirregularity, a deuterated cyclosporin may be administered orally,topically, parenterally, by inhalation spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral, asused herein, includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparation. Tablets containing the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients may alsobe manufactured by known methods. The excipients used may be forexample, (1) inert diluents such as calcium carbonate, lactose, calciumphosphate or sodium phosphate; (2) granulating and disintegrating agentssuch as corn star, or alginic acid; (3) binding agents such as starch,gelatin or acacia, and (4) lubricating agents such as magnesiumstearate, stearic acid or talc. The tablets may be uncoated or the maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay materiel such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets forcontrolled release.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may be

(1) suspending agents such as sodium carboxymethylcellulose,methylcellulose; hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia;

(2) dispersing or wetting agents which may be

-   -   (a) a naturally-occurring phosphatide such as lecithin,    -   (b) a condensation product of an alkylene oxide with a fatty        acid, for example, polyoxyethylene stearate,    -   (c) a condensation product of ethylene oxide with a long chain        aliphatic alcohol, for example, heptadecaethyleneoxycetanol,    -   (d) a condensation product of ethylene oxide with a partial        ester derived from a fatty acid and a hexitol such as        polyoxyethylene sorbitol monooleate, or    -   (e) a condensation product of ethylene oxide with a partial        ester derived from a fatty acid and a hexitol anhydride, for        example polyoxyethylene sorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives, forexample, ethyl or n-propyl p-hydroxybenzoate; one or more coloringagents; one or more flavoring agents; and one or more sweetening agentssuch as sucrose, aspartame or saccharin.

Oily suspension may be formulated by suspending the active ingredient ina vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilsuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide a palatable oral preparation. These compositions may bepreserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in admixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, those sweetening, flavoring and coloring agentsdescribed above may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil such asolive oil or arachis oils, or a mineral oil such as liquid paraffin or amixture thereof. Suitable emulsifying agents may be (1)naturally-occurring gums such as gum acacia and gum tragacanth, (2)naturally-occurring phosphatides such as soy bean and lecithin, (3)esters or partial esters derived from fatty acids and hexitolanhydrides, for example, sorbitan monooleate, (4) condensation productsof said partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol, aspartame or sucrose.Such formulations may also contain a demulcent, a preservative andflavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The disclosed cyclosporines may also be ministered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the disclosed cyclosporines are employed.

Dosage levels of the order from about 0.05 mg to about 50 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (from about 2.5 mg to about 2.5 gms. perpatient per day).

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may containfrom 2.5 mg to 2.5 gm of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Dosage unit forms willgenerally contain between from about 5 mg to about 500 mg of activeingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight generalhealth, sex, diet, time of administration, route of administration, rateof excretion, drug combination and the severity of the particulardisease undergoing therapy.

All references listed herein are incorporated by reference. In the caseof conflict, the text of the application is controlling. Modificationsand changes of the disclosed compounds and methods will be apparent toone skilled in the art. Such modifications and changes are intended tobe encompassed by this disclosure and the claims appended hereto.

1. A cyclosporine A derivative with isotopic or chemical substitution inthe ammo acid position selected from the group consisting of 1, 3 and 9or combinations thereof.
 2. A cyclosporine A derivative according toclaim 1 wherein one or more hydrogen atoms in the amino acid positionselected from the group consisting of 1, 3 and 9 or combinations thereofare substituted with a deuterium atom and wherein said cyclosporine Aderivative is optionally chemically substituted at the amino acid 9position.
 3. The cyclosporin A derivative according to claim 1represented by the formula I:

wherein R is (i) a deuterium or (ii) a saturated or unsaturated straightor branched aliphatic carbon chain of from 1 to 16 carbon atoms andoptionally containing one or more deuterium atoms or an ester, ketone oralcohol of said carbon chain and optionally containing one or moresubstituents selected from halogen, nitro, amino, amido, aromatic, andheterocyclic, or (iii) an aromatic or heterocyclic group conning one ormore deuterium atoms, or (iv) a methyl group and X, Y, and Z arehydrogen or deuterium provided that if R is methyl then at least one ofX, Y or Z is deuterium and wherein R′ is an OH or acetate or other esteror is an O and together with a carbon adjacent to a double bond on aminoacid 1 forms a heterocyclic ring.
 4. The cyclosporine A derivative ofclaim 3 wherein R is a saturated or unsaturated aliphatic carbon chainof from 2 to 3 carbons.
 5. The cyclosporine A derivative of claim 3wherein R is a saturated or unsaturated aliphatic carbon chain of from 2to 3 carbons containing one or more deuterium atoms.
 6. The cyclosporineA derivative of claim 3 wherein R is methyl.
 7. The cyclosporine Aderivative of claim 4 wherein X, Y and Z are hydrogen.
 8. Thecyclosporine A derivative of claim 1 represented by formula 5g:


9. The cyclosporine A derivative of claim 1 represented by formula 5e:


10. A pharmaceutical composition comprising the cyclosporine Aderivative of claim 1 or a pharmaceutically acceptable salt thereof anda pharmaceutically acceptable carrier.
 11. The cyclosporine A derivativeof claim 3 wherein X, Y and Z are H and R is a member selected from thegroup consisting of -D, —CHO, —CDO, —CD₃, —CH═CD-CD₃, —CD=CD-CD₃,—CH═CH—CH═CD-CD₃, —CD=CH—CD=CD-CD₃, —CH═CH—CH═CD₂, —CD=CH—CD=CD₂,—CH-CD₂, —CD-CD₂, —CH—CH₂, —CH═CH—CD₃, —CH═CH—CH₃, —CH═CH—CH═CH—CH₃, and—CH═CH—CH═CH₂.
 12. A method of producing immunosuppression comprising:administering to an animal in need thereof, an effective amount of acyclosporine A derivative according to claim 1.